title: Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system.
abstract: Caspase-3 (CASP3) cleaves many proteins including protein kinases (PKs). Understanding the relationship(s) between CASP3 and its PK substrates is necessary to delineate the apoptosis signaling cascades that are controlled by CASP3 activity. We report herein the characterization of a CASP3-substrate kinome using a simple cell-free system to synthesize a library that contained 304 PKs tagged at their N- and C-termini (NCtagged PKs) and a luminescence assay to report CASP3 cleavage events. Forty-three PKs, including 30 newly identified PKs, were found to be CASP3 substrates, and 28 cleavage sites in 23 PKs were determined. Interestingly, 16 out of the 23 PKs have cleavage sites within 60 residues of their N- or C-termini. Furthermore, 29 of the PKs were cleaved in apoptotic cells, including five that were cleaved near their termini in vitro. In total, approximately 14% of the PKs tested were CASP3 substrates, suggesting that CASP3 cleavage of PKs may be a signature event in apoptotic-signaling cascades. This proteolytic assay method would identify other protease substrates.
authors.affiliation: Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Ehime, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Tadokoro
authors.forename: S
authors.initials: S
authors.lastname: Takahama
authors.forename: K
authors.initials: K
authors.lastname: Shimizu
authors.forename: S
authors.initials: S
authors.lastname: Hayashi
authors.forename: Y
authors.initials: Y
authors.lastname: Endo
authors.forename: T
authors.initials: T
authors.lastname: Sawasaki
mesh_terms: D000818:Animals; D053148:Caspase 3 / Q000378:metabolism*; D002474:Cell-Free System; D006801:Humans; D051379:Mice; D019151:Peptide Library; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D019151:Peptide Library; D011494:Protein Kinases; D053148:Caspase 3